Using genetically engineered chimeric molecules between epidermal growth factor receptor (EGFR) and gp185-erbB-2, this study was undertaken to investigate and characterize the molecular basis of the different biological properties of EGFR and erbB-2 kinases. NIH3T3 cells, overexpressing (NIH-EGFR) or the chimeric EGFR/erbB-2 receptor (NIH- EGFR/erbB-2) containing the extracellular ligand-binding domain of EGFR and the intracellular transduction competent-domain of gp185-erbB-2, were triggered with 50 nM EGF for 15 minutes at 37 degrees C. Phosphotyrosine (pTyr) proteins were immunoprecipitated with anti-pTyr antibodies and analyzed using two-dimensional polyacrylamide gel electrophoresis (2D- PAGE) and anti-pTyr immunoblot analysis. Several proteins were specifically phosphorylated by either EGFR or erbB-2 with markedly different stoichiometry. In NIH3T3 cells, erbB-2 is 100-fold more transforming than EGFR. Concomitant with this transforming capacity is the preferential phosphorylation of at least 28 proteins by erbB-2, as compared to EGFR. To identify which substrates might be directly involved in mitogenic signaling, 2D-PAGE maps of proteins phosphorylated on tyrosine by EGFR/erbB-2-TK, a chimeric molecule in which the tyrosine kinase (TK) domain of erbB-2 replaced that of EGFR, and EGFR/ret, a chimera containing the intracellular portion of ret kinase, a TK belonging to a different family of receptor tyrosine kinases (RTKs), were utilized. Both EGFR/erbB-2-TK and EGFR/ret display an erbB-2-like biological phenotype and transform NIH3T3 with high efficiency. It was anticipated that the use of such chimeras could aid in the identification of specific substrates for erbB-2 mediated mitogenic signaling. In all patterns of pTyr protein expression, paxillin and a 23 kDa substrate were invariably phosphorylated to higher stoichiometry whenever potent mitogenic and transforming signals were activated. It is proposed that paxillin and the 23 kDa substrate are important elements in the erbB-2 and ret-activated mitogenic signaling.